Serum samples collected from 237 dogs in Catalonia (northeastern Spain) were screened by Western blot analysis to detect the presence of antibodies specific to different Leishmania infantum polypeptide fractions. Leishmaniasis was confirmed in 72 of these dogs by direct examination and/or culture. Another 165 animals from the Priorat region were studied periodically for 2-8 years between 1987 and 1995, giving a total of 565 determinations. A control group of 93 dogs from nonendemic areas was also studied. Sera from dogs with leishmaniasis recognized antigens with molecular weights ranging from 12 to 85 kD. The most sensitive antigens were those of 70, 65, 46, 30, 28, 14, and 12 kD, which were recognized by 75%, 75%, 78%, 75%, 81%, 79%, and 75%, respectively, of the sera from dogs with positive parasitologic examination results. Antigens of 70 and 65 kD were also recognized by two dogs from nonendemic areas. Antigens of 14 and 12 kD were the first to be recognized by sera of asymptomatic dogs with titers less than the cut-off value of the dot-ELISA that increased during the longitudinal study, and the presence of antibodies specific for these fractions was observed for up to six years before seroconversion observed by dot-ELISA. These antibodies were also the first to disappear in dogs in which the disease was self-limited. The study corroborates the high sensitivity and specificity of Western blots in the diagnosis of canine leishmaniasis when the bands of low molecular weight (less than 46 kD) are considered, and indicates that fractions of 14 and 12 kD are useful in detecting early forms of the disease.